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Human γD-crystallin, a monomeric protein abundant in the eye lens nucleus, must remain stably folded for an individual’s entire lifetime to avoid aggregation and protein deposition-associated cataract formation. γD-crystallin contains two homologous domains, an N-terminal domain (NTD) and a C-terminal domain (CTD), which interact via a hydrophobic interface. Several familial mutations in the gamma crystallin gene are linked to congenital early-onset cataract, most of which affect the NTD. Some of these, including V75D and W42R, are known to populate intermediates under partially denaturing conditions possessing a natively folded CTD and a completely unfolded NTD. We employed hydrogen–deuterium exchange mass spectrometry to probe the structural and energetic features of variants of γD-crystallin under both native and partially denaturing conditions. For V75D and W42R, we identify a species under native conditions that retains partial structure in the NTD and is structurally and energetically distinct from the intermediate populated under partially denaturing conditions. Residues at the NTD–CTD interface play crucial roles in stabilizing this intermediate, and disruption of interface contacts either by amino acid substitution or partial denaturation permits direct observation of two intermediates simultaneously. These data suggest that the intermediate identified under native conditions is accessed from the native state and not on the folding pathway. The intermediate we have identified here exposes hydrophobic amino acids that are buried in both the folded full-length protein and in the protein’s stable isolated domains. Such nonnative exposure of a hydrophobic patch may play an important role in cataract formation.
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Structural domains of SARS-CoV-2 nucleocapsid protein coordinate to compact long nucleic acid substrates
Abstract The SARS-CoV-2 nucleocapsid (N) protein performs several functions including binding, compacting, and packaging the ∼30 kb viral genome into the viral particle. N protein consists of two ordered domains, with the N terminal domain (NTD) primarily associated with RNA binding and the C terminal domain (CTD) primarily associated with dimerization/oligomerization, and three intrinsically disordered regions, an N-arm, a C-tail, and a linker that connects the NTD and CTD. We utilize an optical tweezers system to isolate a long single-stranded nucleic acid substrate to measure directly the binding and packaging function of N protein at a single molecule level in real time. We find that N protein binds the nucleic acid substrate with high affinity before oligomerizing and forming a highly compact structure. By comparing the activities of truncated protein variants missing the NTD, CTD, and/or linker, we attribute specific steps in this process to the structural domains of N protein, with the NTD driving initial binding to the substrate and ensuring high localized protein density that triggers interprotein interactions mediated by the CTD, which forms a compact and stable protein-nucleic acid complex suitable for packaging into the virion.
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- Award ID(s):
- 1817712
- PAR ID:
- 10438949
- Date Published:
- Journal Name:
- Nucleic Acids Research
- Volume:
- 51
- Issue:
- 1
- ISSN:
- 0305-1048
- Page Range / eLocation ID:
- 290 to 303
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1093/nar/gkac1179
